Characterizing the patterns of clonal selection in circulating tumor DNA from patients with colorectal cancer refractory to anti-EGFR treatment.

INTRODUCTION: KRAS and EGFR ectodomain-acquired mutations in patients with metastatic colorectal cancer (mCRC) have been correlated with acquired resistance to anti-EGFR monoclonal antibodies (mAbs). We investigated the frequency, co-occurrence, and distribution of acquired KRAS and EGFR mutations in patients with mCRC refractory to anti-EGFR mAbs using circulating tumor DNA (ctDNA). PATIENTS AND METHODS: Sixty-two post-treatment plasma and 20 matching pretreatment archival tissue samples from KRAS (wt) mCRC patients refractory to anti-EGFR mAbs were evaluated by high-sensitivity emulsion polymerase chain reaction for KRAS codon 12, 13, 61, and 146 and EGFR 492 mutations. RESULTS: Plasma analyses showed newly detectable EGFR and KRAS mutations in 5/62 [8%; 95% confidence interval (CI) 0.02-0.18] and 27/62 (44%; 95% CI 0.3-0.56) samples, respectively. KRAS codon 61 and 146 mutations were predominant (33% and 11%, respectively), and multiple EGFR and/or KRAS mutations were detected in 11/27 (41%) cases. The percentage of mutant allele reads was inversely correlated with time since last treatment with EGFR mAbs (P = 0.038). In the matching archival tissue, these mutations were detectable as low-allele-frequency clones in 35% of patients with plasma mutations after treatment with anti-EGFR mAbs and correlated with shorter progression-free survival (PFS) compared with the cases with no new mutations (3.0 versus 8.0 months, P = 0.0004). CONCLUSION: Newly detected KRAS and/or EGFR mutations in plasma ctDNA from patients refractory to anti-EGFR treatment appear to derive from rare, pre-existing clones in the primary tumors. These rare clones were associated with shorter PFS in patients receiving anti-EGFR treatment. Multiple simultaneous mutations in KRAS and EGFR in the ctDNA and the decline in allele frequency after discontinuation of anti-EGFR therapy in a subset of patients suggest that several resistance mechanisms can co-exist and that relative clonal burdens may change over time. Monitoring treatment-induced genetic alterations by sequencing ctDNA could identify biomarkers for treatment screening in anti-EGFR-refractory patients.

Clinicopathologic characteristics and gene expression analyses of non-KRAS 12/13, RAS-mutated metastatic colorectal cancer.

BACKGROUND: KRAS mutations in codons 12 and 13 are present in ∼40% of all colorectal cancers (CRC). Activating mutations in codons 61 and 146 of KRAS and in codons 12, 13, and 61 of NRAS also occur but are less frequent. The clinicopathologic features and gene expression profiles of this latter subpopulation of RAS-mutant colorectal tumors have not yet been clearly defined but in general are treated similarly to those with KRAS 12 or 13 mutations. PATIENTS AND METHODS: Records of patients with metastatic CRC (mCRC) treated at MD Anderson Cancer Center between December 2000 and August 2012 were reviewed for RAS (KRAS or NRAS) and BRAF mutation status, clinical characteristics, and survival outcomes. To study further with an independent cohort, data from The Cancer Genome Atlas were analyzed to define a gene expression signature for patients whose tumors feature these atypical RAS mutations and explore differences with KRAS 12/13-mutated colorectal tumors. RESULTS: Among the 484 patients reviewed, KRAS 12/13, KRAS 61/146, NRAS, and BRAF mutations were detected in 47.7%, 3.0%, 4.1%, and 7.4%, respectively, of patients who were tested for each of these aberrations. Lung metastases were more common in both the KRAS 12/13-mutated and atypical RAS-mutated cohorts relative to patients with RAS/BRAF wild-type tumors. Gene expression analyses revealed similar patterns regardless of the site of RAS mutation, and in silico functional algorithms predicted that KRAS and NRAS mutations in codons 12, 13, 61, and 146 alter the protein function and drive tumorgenesis. CONCLUSIONS: Clinicopathologic characteristics, survival outcomes, functional impact, and gene expression profiling were similar between patients with KRAS 12/13 and those with NRAS or KRAS 61/146-mutated mCRC. These clinical and bioinformatic findings support the notion that colorectal tumors driven by these RAS mutations are phenotypically similar.

Interaction between the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF) pathways: a rational approach for multi-target anticancer therapy.

Over the last decade, the concept of targeted biological therapy for the treatment of cancer has emerged. However, a better understanding of these targets and their role in tumor cells and in the surrounding stromal cells is required. Two interesting biological targets are the epidermal growth factor receptor (EGFR) and the vascular endothelia growth factor (VEGF) and its receptors. A number of agents that target these pathways have been described. Many of these are currently in clinical trials and a few have recently been approved by the regulatory authorities in USA and in the European Union. The molecular pathways involved in the proliferation of cancer cells and in tumor-related angiogenesis are very complex and the interference with only a single step of these pathways may often reveal an insufficient therapeutic approach. Moreover, cancer cells have an inherent ability to harness different growth factor signaling pathways for growth advantage and cell survival, a process that may even be facilitated by the use of selective targeted agents. Because of these escape mechanisms, monotherapy with selective targeted agents is unlikely to be a fully effective cancer treatment. For these reasons, targeting different pathways is an attractive and effective therapeutic strategy with a strong rationale for investigating this approach in the clinic. This review focuses on the preclinical rationale of combining targeted agents such as EGFR and VEGF inhibitors in the treatment of cancer and on the clinical trials that have emerged from these studies.

Sequence-dependent antiproliferative effects of cytotoxic drugs and epidermal growth factor receptor inhibitors.

BACKGROUND: Epidermal growth factor receptor (EGFR) inhibitors are in clinical development in cancer treatment. Preclinical studies have shown potential antitumor efficacy of these agents in combination with chemotherapy or with radiotherapy. However, controversial results have been obtained in different clinical trials. MATERIALS AND METHODS: The effects on proliferation, cell cycle distribution and induction of apoptosis of three different anti-EGFR agents (gefitinib, ZD6474, cetuximab) were evaluated in different sequences of combination with either a platinum derivative (cisplatin, carboplatin, oxaliplatin) or a taxane (docetaxel, paclitaxel) in KYSE30 cells, a model of a human cancer cell line with a functional EGFR autocrine pathway. RESULTS: The combination of a cytotoxic drug with an EGFR inhibitor caused different antiproliferative effects on KYSE30 cancer cells depending on the treatment schedule. An antagonistic effect was observed when treatment with each EGFR inhibitor was done before chemotherapy. In contrast, a synergistic antiproliferative activity was obtained when chemotherapy was followed by treatment with EGFR antagonists. This effect was accompanied by potentiation of apoptosis and arrest of the surviving cancer cells in the G(2)/M phases of the cell cycle. CONCLUSIONS: This study provides a rationale for the evaluation of a potentially synergistic sequence of cytotoxic drugs and EGFR inhibitors in a clinical setting.

Effects of butylated hydroxytoluene (BHT) enriched diet on serum antioxidant activity in pre-and overtly diabetic nod mice.

Preventive (antioxidant activity) and chain-breaking (total peroxyl radical-trapping parameter) antioxidants in the serum of controls and butylated hydroxytoluene (BHT)-diet enriched nonobese diabetic (NOD) and C57B16/J mice from 5 to 25 weeks of age are measured in this study. A significant decrease in the overall potency of both antioxidant types is demonstrated in NOD untreated controls but not in animals whose diet was BHT-enriched. Therefore, we show that alterations of the antioxidant status in NOD mice is efficaciously counteracted by BHT.

Synergistic activity of histone deacetylase and proteasome inhibition against pancreatic and hepatocellular cancer cell lines.

AIM: To determine the phenotypic effects of belinostat (bel) and bortezomib (bor) against pancreatic cancer (PC) and hepatocellular cancer (HCC) cell lines. MATERIALS AND METHODS: Antiproliferative effects were assessed using a sulforhodamine B assay. Synergy was evaluated using the Chou and Talalay method. Apoptosis was measured by caspase-3/-7 activity and PARP cleavage. Downstream effector proteins were detected via immunoblotting. Quantitative nuclear magnetic resonance (NMR)-based metabolomics analysis was performed. RESULTS: There were single-agent antiproliferative effects against PC and HCC cell lines; the combination of bel and bor (bel+bor) had a synergistic effect. There was up to a 45-fold induction of apoptosis over the control. Post-treatment cell death was associated with p21 up-regulation, more pronounced with treatment with bel+bor. Treatment with bel+bor enhanced hyperacetylation of histone H3 over single-agent bel. A metabolic signature was established for treatments with bor and bel+bor. CONCLUSION: The combination of bel+bor displayed significant antiproliferative activity against PC and HCC cell lines, with exhibiting synergistic antiproliferative and proapoptotic patterns even at suboptimal single-agent doses.

Small molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer.

Despite recent developments in the diagnosis and conventional treatment of non small cell lung cancer (NSCLC), the prognosis remains unsatisfactory, with 5-year survival rates of approximately 15% for all stages. To date, chemotherapy represents the standard treatment for advanced-non small lung cancer, but efficacy of currently available cytotoxic drugs is modest. Median survival does not exceed 8-10 months. New treatment strategies are needed and considerable hope has been placed in therapies that specifically target the molecular mechanisms of tumour growth. One molecular target of particular relevance to lung cancer pathogenesis is the epidermal growth factor receptor (EGFR), a cell membrane receptor tyrosine kinase. Several inhibitors of EGFR fuctinonal activation have been developed. Amon these, erlotinib (Tarceva) and gefitinib (Iressa) are two orally bioavailable, small molecule EGFR inhibitors of the tyrosine kinase enzymatic activity which prevent EGFR autophosphorylation and activation. In monotherapy, gefitinib and erlotinib have determinated a 10-20% response rate and a 30-50% symptom improvement in previously treated, chemotherapy refractory, advanced NSCLC patients. Furthermore, a randomized, placebo controlled, multicenter phase III study has shown a two months improvement in median survival with erlotinib in the second or third line treatment of metastatic NSCLC patients. We will summarize the clinical evidence on the anticancer activity of small molecule EGFR inhibitors.

Prolactin secretion in polycystic ovary syndrome: circadian rhythmicity and dynamic aspects.

The circadian rhythms of plasma prolactin (PRL) and cortisol and of oral temperature were simultaneously studied in 24 women with polycystic ovary syndrome (PCOS). The PRL response to thyrotropin-releasing hormone (TRH) and domperidone was also evaluated in some of these patients. The physiological circadian chrono-organization of prolactin and cortisol secretion and of oral temperature was maintained in PCOS. The PRL responsiveness to the specific stimulations fell within normal limits. These results do not support the hypothesis of an impaired central dopaminergic regulation of prolactin secretion in PCOS.